The invention relates to protecting groups that can be removed by irradiation.
Protecting groups can be used to mask compounds, or portions of compounds, from interacting in chemical or biological systems. For example, a protecting group can prevent a compound from undergoing a chemical reaction by changing the chemical nature of a functionality. In another example, a protecting group can mask or conceal a biological response induced by the compound in a biological system, both in vivo and in vitro (e.g., in a cell, a tissue, or an assay). One class of protecting group of particular interest for use in biological systems are photolabile protecting groups. Photolabile protecting groups, sometimes called caging groups, have become a mainstay of organic synthesis, biotechnology, and cell biology, because cleavage by light is a very mild deprotection step that is usually orthogonal to other experimental manipulations. The outstanding spatial and temporal precision with which light can be controlled enables diverse applications such as photolithographic construction of complex peptide and oligonucleotide arrays, or physiological release (xe2x80x9cuncagingxe2x80x9d) of bioactive substances in cells and tissues. Examples of caging groups that can be removed from a compound by exposing the compound to light, for example, by a flash of UV, are described in Adams, S. R. and Tsien, R. Y. Annual Rev. Physiology 55:755-784 (1993), incorporated herein by reference.
In general, the protecting groups mask charged (e.g., carboxylate or phosphate) or polar (e.g., amine, sulfhydryl, or hydroxyl) functionalities of the compounds, which can increase their hydrophobicity and their membrane permeability. Before photolysis, these caged compounds are biologically or chemically inactive because at least one of the key functionalities is blocked. The activity of the molecule can be triggered by a pulse of light, which releases the protecting group. In this way, photolabile protecting groups can be removed from a protected compound by irradiation to control release of the compound both spatially and temporally. In particular, compounds of biologically active products can be used to probe biological effects of the compounds. While uncaging can take place in a sample, such as a solution, a tissue sample, or in live cells, this strategy is very valuable for in vivo biological application. It allows control of the onset of bioactivity in living cells with millisecond temporal precision.
Examples of photolabile protecting groups that have been used to cage biomolecules include 2-nitrobenzyl, 1-(2-nitrophenyl)ethyl, 4,5-dimethoxy-2-nitrobenzyl, and -carboxy-4,5-dimethoxy-2-nitrobenzyl. The mechanism of photo-deprotection of caging groups and the applications of caging compounds have been reviewed. See, for example, McCray, J. A. and Trentham, D. R., Annu. Rev. Biophys. Biophys. Chem., 18:239-270 (1989), and Adams, S. R. and Tsien, R. Y., Annu. Rev. Physiol. 55:755-784 (1993). Examples of caged molecules which have had successful applications in biology include caged cAMP (see, e.g., Walker, J. W., et al., Methods Enzymol. 172:288-301 (1989), and Wootton, J. F. and Trentham, D. R., NATO ASI Ser. C 272 (1989)), caged nitric oxide (see, e.g., Lev-Ram, V., et al., Neuron 15:407-415 (1995), and Makings, L. R. and Tsien, R. Y., J. Biol. Chem. 269:6282-6285 (1994)), caged fluorescein (see, e.g., Krafft, G. A., et al., J. Am. Chem. Soc. 110:301 (1988)), caged calcium (see, e.g., Adams, S. R., et al., J. Am. Chem. Soc. 110:3212 (1988), and Tsien, R. Y. and Zucker, R. S., Biophys. J. 50:843-853 (1986)), caged glutamate (see, e.g., Callaway, E. M. and Katz, L. C., Proc. Natl. Acad. Sci. U.S.A. 90:7661-7665 (1993), Wilcox, M., et al., J. Org. Chem. 55:1585 (1990), and Corrie, J. E., et al., J. Physiol. (Lond) 465:1-8 (1993)), and caged inositol-1,4,5-triphosphate (IP3) (see, e.g., Walker, J. W., et al., Nature 327:249-252 (1987)).
In general, the invention features a protecting group derived from a halogenated coumarin group, a quinoline-2-one group, a xanthene group, a thioxanthene group, a selenoxanthene group, or an anthracene group. The protecting group is photolabile and can be removed by irradiating the group with light, such as flash photolysis with ultraviolet radiation or pulsed infrared radiation.
In one aspect, the invention features a protecting group derived from a halogenated coumarin or quinoline-2-one group. The protecting group can be a part of a compound. The compound has the formula: 
In the formula: A is xe2x80x94OH, substituted or unsubstituted alkoxy, xe2x80x94OC(O)CH3, xe2x80x94NH2, or xe2x80x94NHCH3; each of X1 and X2, independently, is H, Cl, Br, or I, at least one of X1 and X2 being Cl, Br, or I, Q is xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, or xe2x80x94NCH3xe2x80x94; Y1 is xe2x80x94H, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94C(O)OH, xe2x80x94NO2, xe2x80x94C(O)NHR1, xe2x80x94CN, xe2x80x94C(O)H, xe2x80x94C(O)CH3, benzoxazol-2-yl, benzothiazol-2-yl, or benzimidazol-2-yl; Y2 is xe2x80x94H, xe2x80x94C(O)OH, or xe2x80x94SO3H; M1 is xe2x80x94H, xe2x80x94CH3, xe2x80x94NR2R3, xe2x80x94C(O)NR2R3, or xe2x80x94COOH; Z is a leaving group and M2 is xe2x80x94H, or Z and M2 together are xe2x95x90N2, xe2x95x90O, or xe2x95x90NNHR1; and each of R1, R2, and R3, independently, is a substituted or unsubstituted moiety selected from the group consisting of a C1-20 alkyl, a C2-20 alkenyl, a C2-20 alkynyl, a C1-20 alkoxy, a C1-20 thioalkoxy, a C1-20 alkylsulfonyl, a C4-16 arylsulfonyl, a C2-20 heteroalkyl, a C2-20 heteroalkenyl, a C3-8 cycloalkyl, a C3-8 cycloalkenyl, a C4-16 aryl, a C4-16 heteroaryl, and a C2-30 heterocyclyl. The compound can be a salt.
In another aspect, the invention features a protecting group derived from a xanthene group, a thioxanthene group, a selenoxanthene group, or an anthracene group. The protecting group can be a part of a compound. The compound has the formula: 
In the formula: E1 is xe2x80x94OH, substituted or unsubstituted alkoxy, xe2x80x94OC(O)CH3, xe2x80x94NH2, xe2x80x94NHCH3, or xe2x80x94N(CH3)2; E2 is xe2x95x90O, xe2x95x90NH2+, xe2x95x90NHCH3+, or xe2x95x90N(CH3)2+; G is O, S, SO2, Se, or C(CH3)2; each of J1, J2, J3, and J4, independently, is H, F, Cl, Br, or I; each of L1 and L2, independently, is H, xe2x80x94C(O)OH, or xe2x80x94SO3H; M1 is xe2x80x94H, xe2x80x94CH3, substituted amino, disubstituted amino, amido, xe2x80x94COOH, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 heteroalkyl, or substituted or unsubstituted C2-30 heterocyclyl; Z is a leaving group and M2 is xe2x80x94H, or Z and M2 together are xe2x95x90N2, xe2x95x90O, or xe2x95x90NNHR1; and R1 is a substituted or unsubstituted moiety selected from the group consisting of a C1-20 alkyl, a C2-20 alkenyl, a C2-20 alkynyl, a C1-20 alkoxy, a C1-20 thioalkoxy, a C1-20 alkylsulfonyl, a C4-16 arylsulfonyl, a C2-20 heteroalkyl, a C2-20 heteroalkenyl, a C3-8 cycloalkyl, a C3-8 cycloalkenyl, a C4-16 aryl, a C4-16 heteroaryl, and a C2-30 heterocyclyl. The compound can be a salt.
In preferred embodiments, Z and M2 together are xe2x95x90N2, xe2x95x90O, or xe2x95x90NNHR1. In other preferred embodiments, Z is a leaving group and M2 is xe2x80x94H.
A leaving group is a group that can be photolytically displaced. Generally, a leaving group departs from a substrate with the pair of electrons of the covalent bond between the leaving group and the substrate. Preferred leaving groups stabilize the pair of electrons via the presence of electron withdrawing groups, aromaticity, resonance structures, or a combination thereof. Examples of leaving groups include halide, or moieties linked by a carboxylate, a carbonate, an amide, a carbamate, a phosphate, a sulfonate, an amino, an aryloxide, or a thiolate group.
When Z is a leaving group, Z can be a halogen, xe2x80x94OC(O)R4, xe2x80x94OP(O)R5R6, xe2x80x94OP(O)(OH) R5, xe2x80x94OC(O)NR5R6, xe2x80x94NR5C(O)OR6, xe2x80x94SR4, alkoxy, aryloxy, xe2x80x94NR5R5, xe2x80x94NR5C(O)R6, xe2x80x94O3SR4, or xe2x80x94Oxe2x80x94NN(O)(NR5R6). Each of R4, R5, and R6, independently, can be a substituted or unsubstituted moiety selected from the group consisting of a C1-20 alkyl, a C2-20 alkenyl, a C2-20 alkynyl, a C1-20 alkoxy, a C1-20 thioalkoxy, a C1-20 alkylsulfonyl, a C4-16 arylsulfonyl, a C2-20 heteroalkyl, a C2-20 heteroalkenyl, a C3-8 cycloalkyl, a C3-8 cycloalkenyl, a C4-16 aryl, a C4-16 heteroaryl, a C2-30 heterocyclyl, a cyclitol radical, a saccharide radical, a saccharide phosphate radical, a polysaccharide radical, a lipid radical, an amino acid radical, a peptide radical, a nucleoside radical, a nucleotide radical, a nucleoside monophosphate radical, a nucleoside thiophosphate radical, a nucleoside triphosphate radical, a nucleoside diphosphate radical, and a polynucleotide radical. Alternatively, R5 and R6, together, can form a substituted or unsubstituted moiety selected from the group consisting of a C1-20 alkylene, a cyclitol diradical, a saccharide diradical, a saccharide phosphate diradical, a polysaccharide diradical, a lipid diradical, an amino acid diradical, a peptide diradical, a nucleoside diradical, a nucleotide diradical, a nucleoside monophosphate diradical, a nucleoside thiophosphate diradical, a nucleoside diphosphate diradical, a nucleoside triphosphate diradical, or a polynucleotide diradical.
In preferred embodiments, when the protecting group is a derivative of a halogenated coumarin, Q is O, A is xe2x80x94OH, xe2x80x94OCH3, or xe2x80x94OC2H5, X2 is xe2x80x94H, Y2 is xe2x80x94H, Y1 is xe2x80x94H, Z is xe2x80x94Cl, xe2x80x94OC(O)O-(4-nitrophenyl), xe2x80x94NR5C(O)R6, xe2x80x94OC(O)NR5R6, or xe2x80x94OP(O)R5R6. In certain embodiments, R5 can be H and R6 can be an amino acid radical or a peptide radical, or R5 and R6, together, can form a nucleoside diradical.
In other preferred embodiments, when the protecting group is a xanthene derivative, E2 is xe2x95x90O, G is O, and M1 is H, each of J1, J2, J3, and J4 is H, M2 is H, and Z is xe2x80x94Cl, xe2x80x94OC(O)R1, or xe2x80x94OC(O)O-(4-nitrophenyl).
In another aspect, the invention features a method of deprotecting a compound. The method includes exposing a caged compound having a photolabile protecting group for a sufficient time to remove the photolabile protecting group and deprotect the compound. The caged compound has a two photon action cross section at 740 nm of at least 0.1xc3x9710xe2x88x9250 cm4s.
In another aspect, the invention features a method of deprotecting a compound. The method includes exposing a caged compound having a photolabile protecting group derived from a derived from a halogenated coumarin, a quinoline-2-one, a xanthene, a thioxanthene, a selenoxanthene, or an anthracene to a sufficient amount of radiation for a sufficient time to remove the photolabile protecting group and deprotect the compound.
In another aspect, the invention features a method for introducing a compound into a sample. The method includes the steps of contacting the sample with a caged compound having a photolabile protecting group derived from a halogenated coumarin, a quinoline-2-one, a xanthene, a thioxanthene, a selenoxanthene, or an anthracene, and exposing the caged compound to a sufficient amount of radiation for a sufficient time to remove the photolabile protecting group and deprotect the compound.
In another aspect, the invention features a method for introducing a compound into a sample. The method includes the steps of contacting the sample with a caged compound having a photolabile protecting group, and exposing the caged compound to a sufficient amount of radiation for a sufficient time to remove the photolabile protecting group and deprotect the compound. The caged compound has a two photon action cross section at 740 nm of at least 0.1xc3x9710xe2x88x9250 cm4s.
The sample can be a biological sample, a spatially arrayed combinatorial library, or an optical memory (e.g., a three dimensional optical memory).
The compound can have a one photon action cross section at 365 nm of at least 110 Mxe2x88x921cmxe2x88x921 (e.g., at least 200 Mxe2x88x921cmxe2x88x921, preferably at least 300 Mxe2x88x921cmxe2x88x921) . The compound can have a two photon action cross section at 740 nm of at least 0.1xc3x9710xe2x88x9250 cm4s/photon (e.g., at least 0.5xc3x9710xe2x88x9250 cm4s/photon, preferably at least 1.0xc3x9710xe2x88x9250 cm4s/photon). The compound can have a two photon action cross section at 800 nm of at least 0.05xc3x9710xe2x88x9250 cm4s/photon (e.g., at least 0.1xc3x9710xe2x88x9250 cm4s/photon, preferably at least 0.2xc3x9710xe2x88x9250 cm4s/photon).
The radiation can be ultraviolet radiation or infrared radiation (e.g., pulsed infrared radiation). The exposing step can be a two photon process.
The compound can have the formula Z-H. Z can be halogen, xe2x80x94OC(O)R4, xe2x80x94OP(O)R5R6, xe2x80x94OP(O)(OH)R5, xe2x80x94OC(O)NR5R6, xe2x80x94NR5C(O)OR6, xe2x80x94SR4, alkoxy, aryloxy, xe2x80x94NR5R5, xe2x80x94NR5C(O)R6, or xe2x80x94O3SR4; and each of R4, R5, and R6, independently, is a substituted or unsubstituted moiety selected from the group consisting of a C1-20 alkyl, a C2-20 alkenyl, a C2-20 alkynyl, a C1-20 alkoxy, a C1-20 thioalkoxy, a C2-20 heteroalkyl, a C2-20 heteroalkenyl, a C3-8 cycloalkyl, a C3-8 cycloalkenyl, a C4-16 aryl, a C4-16 heteroaryl, a C2-30 heterocyclyl, a cyclitol radical, a saccharide radical, a saccharide phosphate radical, a polysaccharide radical, a lipid radical, an amino acid radical, a peptide radical, a nucleoside radical, a nucleotide radical, a nucleoside monophosphate radical, a nucleoside thiophosphate radical, a nucleoside diphosphate radical, a nucleoside triphosphate radical, and a polynucleotide radical; or R5 and R6, together, form a substituted or unsubstituted moiety selected from the group consisting of a C1-20 alkylene, a cyclitol diradical, a saccharide diradical, a saccharide phosphate diradical, a polysaccharide diradical, a lipid diradical, an amino acid diradical, a peptide diradical, a nucleoside diradical, a nucleotide diradical, a nucleoside monophosphate diradical, a nucleoside thiophosphate diradical, a nucleoside diphosphate diradical, a nucleoside diphosphate diradical or a polynucleotide diradical. The compound can be a salt.
The photolabile protecting group can have the formula 
in which A is xe2x80x94OH, substituted or unsubstituted alkoxy, xe2x80x94OC(O)CH3, xe2x80x94NH2, or xe2x80x94NHCH3; each of X1 and X2, independently, is H, Cl, Br, or I, at least one of X1 and X2 being Cl, Br, or I; Q is xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, or xe2x80x94NCH3xe2x80x94; Y1 is xe2x80x94H, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94C(O)OH, xe2x80x94NO2, xe2x80x94C(O)NHR1, xe2x80x94CN, xe2x80x94C(O)H, xe2x80x94C(O)CH3, benzoxazol-2-yl, benzothiazol-2-yl, or benzimidazol-2-yl; Y2 is xe2x80x94H, xe2x80x94C(O)OH, or xe2x80x94SO3H; M1 is xe2x80x94H, xe2x80x94CH3, xe2x80x94NR2R3, xe2x80x94C(O)NR2R3, or xe2x80x94COOH; and each of R1, R2, and R3, independently, is a substituted or unsubstituted moiety selected from the group consisting of a C1-20 alkyl, a C2-20 alkenyl, a C2-20 alkynyl, a C1-20 alkoxy, a C1-20 thioalkoxy, a C1-20 alkylsulfonyl, a C4-16 arylsulfonyl, a C2-20 heteroalkyl, a C2-20 heteroalkenyl, a C3-8 cycloalkyl, a C3-8 cycloalkenyl, a C4-16 aryl, a C4-16 heteroaryl, and a C2-30 heterocyclyl.
Alternatively, the photolabile protecting group can have the formula 
in which E1 is xe2x80x94OH, xe2x80x94OCH3, xe2x80x94OC2H5, xe2x80x94OC(O)CH3, xe2x80x94NH2, xe2x80x94NHCH3, or xe2x80x94N(CH3)2; E2 is xe2x95x90O, xe2x95x90NH2+, xe2x95x90NHCH3+, or xe2x95x90N(CH3)2+; G is O, S, Se, SO2, or C(CH3)2; each of J1, J2, J3, and J2, independently, is H, F, Cl, Br, or I; each of L1 and L2, independently, is H, xe2x80x94C(O)OH, or xe2x80x94SO3H; and M1 is xe2x80x94H, xe2x80x94CH3, substituted amino, disubstituted amino, amido, xe2x80x94COOH, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 heteroalkyl, or substituted or unsubstituted C2-30 heterocyclyl.
Each R1, R2, R3, R4, R5, or R6 group can be substituted with one or more substituent groups. Substituted groups may have one, two, three or more substituents, which may be the same or different, each replacing a hydrogen atom. Substituents include halogen (e.g., F, Cl, Br, and I), hydroxyl, protected hydroxyl, amino (e.g., alkyl amino, dialkyl amino, or trialkyl ammonium), protected amino, carboxy, protected carboxy, cyano, methylsulfonylamino, alkoxy, acyloxy, nitro, sulfhydryl, phosphate, aryl groups, and lower haloalkyl. The substituent can also be a polymer, such as a functionalized polystyrene connected by a linking group to the photolabile protecting group.
An amino acid group is a moiety having an amino group and a carboxylic acid group, such as an xcex1-amino acid. An amino acid includes the 20 common xcex1-amino acids (Gly, Ala, Val, Leu, Ile, Ser, Thr, Asp, Asn, Lys, Glu, Gln, Arg, His, Phe, Cys, Trp, Tyr, Met and Pro), and other amino acids that are natural products, such as norleucine, ethylglycine, ornithine, gamma-amino butyric acid, and phenylglycine. A peptide group is composed of two or more amino acid groups linked by an amide bond.
A radical is a group of atoms that is bonded to moiety via a single bond through a single atom of the group. A diradical is a group of atoms that is bonded to moiety via two single bonds through two different atoms of the group.
An alkyl group is a branched or unbranched hydrocarbon that may be substituted or unsubstituted. Examples of branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl, tert-pentyl, isohexyl. An alkenyl group contains one or more carbon-carbon double bonds. An alkynyl group contains one or more carbon-carbon triple bonds. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. An aryl group is an aromatic ring, where the ring is made of carbon atoms. A cycloalkenyl group is a cycloalkyl containing a carbon-carbon double bond. A cyclitol radical is a cycloalkyl group having one or more hydroxyl groups (e.g., inositol).
An alkoxy group is an alkyl group linked to an oxygen atom through which it is linked to another moiety. When the leaving group is an alkoxy group, the alkoxide anion can be the conjugate base of an alcohol having a low pH. Examples of suitable alkoxy groups include xe2x80x94OCCl3, and xe2x80x94OCF3. An aryloxy group is an aryl group linked to an oxygen atom through which it is linked to another moiety. A thioalkoxy is an alkyl group linked to a sulfur atom through which it is linked to another moiety. An alkylsulfonyl or arylsulfonyl group is an alkyl or aryl group linked to a sulfonyl group through which it is linked to another moiety.
A heteroalkyl, a heteroalkenyl, heterocyclyl group contains at least one ring structure which contains carbon atoms and at least one heteroatom (e.g., N, O, S, or P). A heteroaryl is an aromatic heterocyclic radical. Examples of heterocyclyl radicals and heteroaryl groups include: thiazolyl, thienyl, furyl, 1-isobenzofuranyl, 2H-chromen-3-yl, 2H-pyrrolyl, N-pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, phthalazinyl, cinnolinyl, benzoxazol-2-yl, benzothiazol-2-yl, or benzimidazol-2-yl, and pteridinyl. A heterocyclyl group may be attached to another moiety via a carbon atom or a heteroatom of the heterocyclic radical.
A saccharide group is a radical of a saccharide. A saccharide is a compound having the general formula (CH2O)n, including hexoses (n=6) and pentoses (n=5). A saccharide phosphate group is a saccharide radical bonded to at least one phosphate group. A polysaccharide group is a polymer composed of two or more saccharide groups bonded to each other. A lipid group is a radical derived from a lipid, which is composed of three fatty acid chains linked to a glycerol backbone by ester linkages. A nucleoside group is a radical including a base (e.g., a purine or a pyrimidine) linked to a saccharide group or a deoxy-saccharide group (e.g., ribose or deoxyribose, respectively). A nucleotide group is a nucleoside group linked to one or more phosphate moiety. A nucleoside monophosphate group is a nucleotide having a single phosphate moiety. A nucleoside diphosphate group is a nucleotide having a two phosphate moieties. A nucleoside triphosphate group is a nucleotide having a three phosphate moieties. A nucleoside thiophosphate group is a nucleotide in which at least one oxygen atom of a phosphate group is replaced by a sulfur atom. A polynucleotide group is a polymer composed of two or more nucleotide groups bonded to each other.
The caging group can interfere with the binding, reactivity, or activity of the compound. For example, caged amino acids can be used in the automated synthesis of peptides that may be biologically inactive until photolyzed. Amino acids caged on the xcex1-amine can block the N-terminus of the peptide during synthesis.
In another example, caged compounds can also be used to probe the effects of the uncaged compounds in a biological sample. Biological samples can include muscle fibers, muscle cells, brain tissue, brain cells, fibroblasts, sarcoplasmic reticulum vesicles, submitochondrial particles, membrane fragments, samples containing regulatory proteins. In addition, caged chelants can bind metal ions, for example, Ca2+ or Mg2+. The affinity of the chelant for a particular metal ion can change when the protecting group is removed by photolysis. This change in affinity can lead to a rapid and localized change (e.g., increase or decrease) in metal ion concentration, which can permit changes in localized metal ion concentration changes to be studied.
The halogenated coumarin- and xanthene-based caging groups attached to the leaving group via a xe2x80x94CHM1-group (e.g., a CH2 group) to a site that undergoes a large increase in electron density upon excitation of the delocalized chromophore. The halogen atoms of the coumarin-based group and the extended conjugation of the xanthene based group are added to promote intersystem crossing to the triplet state. These design principles can be applied to other fluorescent dyes to create other caging groups of longer wavelengths and larger one photon and two photon cross-sections. For use in one photon photolysis, longer wavelengths and greater photosensitivity can help minimize photodamage, decrease irradiation times, simplify experimental apparatus, make semiconductor light sources feasible for uncaging, and permit separate and controllable photolyses of two or more protecting groups at different wavelengths.
The two-photon photolysis can use less energy and be less damaging to the surroundings of the compound being uncaged. A high degree of three-dimensional spatial precision can be obtained by excitation with two or more coincident infrared photons of equivalent total energy. Such multiphoton excitation can require extremely high local intensities, typically obtained by focusing a femtosecond pulsed infrared (IR) laser with a high-numerical-aperture lens, and becomes insignificant away from the point of focus. This nonlinear optical phenomenon can be used to noninvasively localize the photochemistry to any given spot in three dimensions and can be especially valuable in mapping biochemical sensitivities in complex tissues such as the brain. Photosensitivity is quantified as the uncaging action cross-section xcex4u, which is the product of the two-photon absorbance cross-section xcex4a and the uncaging quantum yield Qu2. Ideally, xcex4u should exceed 0.1 GM, where GM or Goeppert-Mayer is defined as 10xe2x88x9250 cm4.s/photon. Carboxylate, phosphate, and carbamate esters of brominated 6-hydroxycoumarin-4-ylmethanol have the requisite action cross-sections (xcex4uxcx9c1 GM), photolysis kinetics, synthetic accessibility, water solubility, and stability in the dark to be used in the true two-photon uncaging of biologically important acids and amines. In addition, their cross-sections for one-photon uncaging with UV radiation at 365 nm or longer are high. Two-photon photolysis can lead to higher-resolution three-dimensional optical memories, spatially arrayed combinatorial libraries, photodynamic therapy, and deeper and less-invasive mapping of the local responses of complex tissues to neurotransmitters and messengers.
Other features or advantages of the present invention will be apparent from the following detailed description and from the claims.